Kit and method for rapidly and visually detecting PRRSV based on Cas12a protein
A technology of PRRSV-RPA-F and PRRSV-RPA-R, which is applied in the field of molecular biology, can solve the problems of high detection conditions, inability to detect quickly, expensive detection instruments, etc., and achieve accurate detection results, important economic benefits and social benefits. The effect of high value and high detection rate
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[0111] According to a preferred embodiment of the invention, the CrRNA sequence specifically identified by the NSP2 gene is selected from one or more of CrRNA-SGRNA1 to CrNA-SGRNA50.
[0112] Preferably, the sequence of the CrRNA-SGRNA1 to CrNA-SGRNA50 is specifically shown below:
[0113] CrRNA-SGRNA1: 5'-uuuncccaucgucaaccgaugg aucuacaacaguagaaau-3 ';
[0114] CrRNA-SGRNA2: 5'-uuunagacccccuuccugaaagaaucuacaacaguagaaaau-3 ';
[0115] CrRNA-SGRNA3: 5'-uuuncucccuugaauguucaaucuacaacaguagaaau-3 ';
[0116] CrRNA-SGRNA4: 5'-uuunagcacguacuuggcccccaccaucuacaacaguagaaau-3 ';
[0117] CrRNA-SGRNA5: 5'-uuungauguuguucacaagauccuaucuacaacaguagaaau-3 ';
[0118] CrRNA-SGRNA6: 5'-uuunaggaaggoggucucaucucaaguaucuacaacaguagaaau-3 ';
[0119] CrRNA-SGRNA7: 5'-uuuncucccuugaauguucaucuacaacaguagaaau-3 ';
[0120] CrRNA-SGRNA8: 5'-uuunnuccccuugaauguucaaucuacaacaguagaaau-3 ';
[0121] CrRNA-SGRNA9: 5'-uuuncnccccuugaauguucaaucuacaacaguagaaau-3 ';
[0122] CrRNA-SGRNA10: 5'-uuuncuncccuugaauguucaucuacaacagu...
Embodiment 1
[0229] 1, test samples
[0230] a, randomly selected channel in Huaihua city phthalocyanine farming cooperatives Wu (variety: A Dual hetero (Landrace and Large White), gender: female) pig spleen tissue sample;
[0231] B, a plasmid carrying the sample information PRRSV conserved viral sequences, such as nucleotide sequence SEQ ID NO: 3 shown in FIG.
[0232] 2, extracted and purified RNA a sample
[0233] 8 parts collected pig spleen tissue, total RNA was extracted for different individuals;
[0234] RNA isolation perform the following steps:
[0235] Organization (2.1) picked up with tweezers fresh spleen tissue, and the bean-sized clipped with scissors into a clean an EP tube, minced tissue with small scissors, then quickly placed 1000μl TRNzol solution;
[0236] (2.2) was added to a mixture of 5 tissues in diameter and 2mm TRNzol solution for the ball, when the amplitude 50Hz / min, the polishing shaken three times 90s, take it out to see tissue ablation substantially without c...
experiment example 1
[0280] In Example 1 Sample B (the plasmid carrying the viral conserved sequence information sample PRRSV) is serially diluted, 10 respectively 10 10 9 10 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 10 0 A PRRSV-specific fragment copies, using the method described in Example 1 and the conventional method of the above-described conventional PCR 11 copies of PRRSV-specific fragments were detected, as were the results figure 1 and 2 Indicated.
[0281] Wherein, RPA amplification reaction with water as a negative control in (the NC);
[0282] PCR amplification detection system common method is: 94 ℃ 5min; 35 cycles: 94 ℃ 30s, 54 ℃ 30s, 72 ℃ 20s; 72 ℃ 7min final extension.
[0283] Specific detection system as follows:
[0284] Detection Primer:
[0285] nsp2-F: CTACTCTCCGCCTGCCGA (such as SEQ ID NO: 5 shown)
[0286] nsp2-R: CCTGAACACATTCAAG (such as SEQ ID NO: 6 below)
[0287] Reagent concentration Volume (μL) 2X Mix NA 25 Hide 2 O
NA 20 Nsp2-f 10um 2 Nsp2...
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